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Regular Articles |
From the Departments of Immunology*
and
Cancer Biology,
University of Texas M. D.
Anderson Cancer Center, Houston, Texas; and the Department of
Pathology,
University of Tasmania, Hobart,
Tasmania, Australia
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The p53 tumor suppressor gene is considered the guardian of the genome14 and is one of the most frequently mutated genes in UV-induced human and mouse skin cancers.15-17 DNA-damaging agents such as UV and ionizing radiation induce high levels of p53,18-27 which in turn activates the transcription of downstream genes responsible for cell cycle arrest at the G1-S transition.28 The G1-S arrest results, at least in part, from p53 transactivation of p21Waf-1/Cip1, which binds to and inactivates the cyclin-dependent kinases required for cell cycle progression.29-31 This growth arrest allows the cells to repair the DNA damage.32-34 However, p53 can also cause apoptosis of cells with excessive unrepaired DNA damage35-36 by activation of bax and/or down-regulation of bcl-2 expression.37-39
In vitro and in vivo studies from different laboratories have shown that UV induces the expression of p5318-27,40 and p21Waf-1/Cip122,25,29-31 and the formation of sunburn cells.36 However, the effects of UV radiation on the time course for induction of p53 and its downstream effectors, formation of sunburn (apoptotic) cells, changes in expression of apoptosis-regulatory molecules, and hyperplasia have not been investigated in the same system. This is quite important to understanding the temporal events, at the cellular and molecular level, in skin injury (sunburn) caused by exposure to UV radiation and the adaptive responses that the skin employs to cope with this injury. In the study described here, we examined the time course for induction of sunburn cells and epidermal hyperplasia in UV-irradiated mouse skin. In addition, we investigated the temporal changes in expression, not only of proteins known to be associated with growth arrest such as p53 and p21Waf-1/Cip1, but also those associated with apoptosis, bax and bcl-2, and proliferation, proliferating cell nuclear antigen (PCNA).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female SKH-hr1 mice 7 to 8 weeks old were obtained from Charles River (Wilmington, MA) and housed in cages in a room with controlled temperature and humidity and alternating 12-hour light and dark cycles. The room was lit with yellow fluorescent lamps (Mazda S.10) in ceiling fixtures with plastic diffusers to eliminate all ambient UV radiation. The mice were fed with a commercial diet and had water ad libitum.
Twenty-one mice were used in this experiment. One group of 3 mice was
not exposed to UV and was used as a control. The other 18 mice were
placed into standard cages (
5 mice/cage) separated into
individual compartments with Plexiglas dividers. The animal cages were
placed on a shelf 20 cm below the light source, and mice were
irradiated with a single dose of 2.5 kJ/m2
UV
from a bank of six Kodacel-filtered (290–400 nm) FS40 sunlamps, as
described previously.41
The irradiance of the sunlamps was
measured with an IL-700 radiometer with an SEE 240 UVB detector
equipped with an A127 quartz diffuser (International Light,
Newburyport, MA). The UVB detector was placed in a cage with a wire top
under the lights, and the UVB was measured. Under this protocol the
mice were exposed to 2.5 W/m2
UVB radiation.
Isolation of Skin Samples
Groups of 3 mice were killed at 3, 6, 12, 24, 48, and 72 hours after UV irradiation. The dorsal skin (approximately 2 x 4 cm) was excised from each mouse and cut into 2 pieces. One piece was immediately fixed in 4% buffered formaldehyde for paraffin embedding. The other piece was floated dermis side down on 0.5 mol/L buffered EDTA solution, pH 7.4, for 1 hour at 37°C, and the epidermis was separated from the dermis. Cell lysates were prepared from the epidermis in the cold (on ice) and immediately frozen at -80°C. The paraffin-embedded skin was cut into 5-µm sections, deparaffinized, hydrated, dehydrated, and stained with hematoxylin and eosin.
Terminal Deoxynucleotidyl Transferase Nick End Labeling (TUNEL) Assay
In addition to defining the sunburn cells histologically, UV-irradiated mouse skin was examined for the presence of apoptotic cells by the TUNEL42 assay. This assay provides a relatively reliable measure of apoptosis and readily identifies fragmented DNA. The TUNEL assay was performed using a commercial kit according to the manufacturer’s protocol (Promega Corp., Madison, WI). Briefly, the 5-µm sections were deparaffinized and fixed in 4% paraformaldehyde at room temperature for 5 minutes. Then, they were treated with 20 µg/ml proteinase K for 10 minutes and permeabilized by incubation with 0.5% Triton X-100 in phosphate-buffered saline (PBS) for 5 minutes at room temperature. After being rinsed twice with PBS for 5 minutes, the slides were incubated with reaction buffer containing terminal deoxynucleotidyl transferase and fluorescein-12-dUTP in a humid atmosphere at 37°C for 1 hour. EDTA was added to the slides for 5 minutes to stop the reaction, and the slides were washed three times with PBS for 5 minutes and stained with 10 µg/ml propidium iodide for 10 minutes. Finally, after 3 washes with PBS for 5 minutes, coverslips were mounted with Prolong reagent (Molecular Probes, Eugene, OR) to prevent fluorescence bleaching during analysis, and fragmented DNA was identified by measurement of incorporated fluorescein-12-dUTP. The slides were examined with an Olympus Inverted System Microscope IX70 (Olympus, Melville, NY), and pictures were taken with a Nikon 35-mm camera. The number of TUNEL-positive cells per 100 cells in a field were counted under a microscope. Four such fields were counted for each of the three mice used for each time point. The mean values and standard deviations were calculated using StatView 4.0.
Western Blot Analysis
The epidermis of each mouse was homogenized, and proteins were
extracted with a lysis buffer (120 mmol/L NaCl, 25 mmol/L Tris, pH 7.5,
and 1% Triton X-100) containing protease inhibitor cocktail
(Boehringer Mannheim, Indianapolis, IN) for 1 hour on ice. Equal
amounts of protein extract (100 µg) from each lysate were
electrophoresed on 12% polyacrylamide-sodium dodecyl sulfate gels,
transferred onto a nitrocellulose membrane, and incubated with specific
antibodies. The antibodies used were mouse anti-p53 monoclonal antibody
PAb240 (cat. no. NCL-p53–240, NovoCastra, Newcastle, UK), monoclonal
anti-p21Waf1/Cip1 antibody Ab-4 (cat. no. OP76,
Oncogene Science, Inc., Uniondale, NY), monoclonal anti-bcl-2 antibody
(cat. no. 15616E, Pharmingen, San Diego, CA), rabbit polyclonal
anti-mouse bax antibody (cat. no. sc-493, Santa Cruz Biotechnology,
Santa Cruz, CA) and mouse anti-human PCNA antibody clone PC10 (cat. no.
M0879, DAKO, Carpenteria, CA) diluted 1:100 in PBS. Only p53 proteins
were initially immunoprecipitated with anti-p53 monoclonal antibody
Ab-3 (cat. no. OP29, Oncogene Science) before the electrophoresis.
After incubation with the appropriate secondary antibody, signals were
detected using immunochemiluminescent reagents (Amersham Life Science,
Poole, UK) and autoradiography. Equal protein loading in each
lane was confirmed by hybridization with a 1:2000 dilution of 
-actin
antibody (Sigma Chemical Co., St. Louis, MO). The band intensities were
quantified by densitometric scanning of autoradiographs using Image
Quant (Molecular Dynamics, Sunnyvale, CA). Three optical density
values, corresponding to the three mice used per each time point, were
normalized for -actin and then averaged. The resulting mean values
were compared with the control (unirradiated) skin to determine the
increase or decrease of protein expression level. Statistical analysis
was performed using StatView 4.0.
Immunohistochemical Analysis
Immunohistochemical assays were performed according to Berg et al,43 with slight modifications. After deparaffinization, 5-µm sections were treated with target retrieval solution (DAKO), washed 3 times with PBS and incubated in H2O2/methanol/PBS solution (1:50:50) for 15 minutes to block endogenous peroxidase activity. After 3 washes in PBS with 0.5% Tween, the sections were preincubated for 10 minutes in 10% normal goat serum in PBS and then incubated with the first antibody overnight at 4°C. The antibodies used were: (i) rabbit polyclonal anti-mouse p53 antibody (cat. no. NCLp53-CM5, NovoCastra) diluted 1:1000 in PBS, (ii) rabbit polyclonal anti-mouse p21 antibody (cat. no. sc-471, Santa Cruz Biotechnology) diluted 1:500 in PBS, (iii) rabbit polyclonal anti-mouse bcl-2 antibody (cat. no. 15021A, Pharmingen) diluted 1:500 in PBS, (iv) rabbit polyclonal anti-mouse bax antibody (cat. no. sc-493, Santa Cruz Biotechnology,) diluted 1:500 in PBS, and (v) mouse anti-human PCNA antibody clone PC10 (cat. no. M0879, DAKO) diluted 1:50 in PBS. After 3 washes in PBS plus 0.5% Tween, the sections were incubated for 1 hour at room temperature with the following secondary antibodies: (i) biotin-conjugated goat anti-rabbit antibody solution for p53 (Vector Laboratories, Burlingame, CA), (ii) donkey anti-rabbit horseradish peroxidase-linked F(ab)2 fragment (cat. no. NA9340, Amersham Life Science) diluted 1:100 in PBS for p21, bcl-2, and bax, and (iii) anti-mouse horseradish peroxidase-linked antibody (cat. no. NA931, Amersham Life Science) diluted 1:100 in PBS for PCNA. After a wash in PBS, p53 staining was performed with the Vectastain Elite ABC kit with diaminobenzidine as the chromagen, as recommended by the manufacturer (Vector Laboratories) or by direct diaminobenzidine staining for p21, bcl-2, bax, and PCNA. Counterstaining was performed with hematoxylin. As a negative control, tissue sections were stained only with the secondary antibody.
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Results |
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UV-irradiated mouse skin was examined for the presence of sunburn
cells at various times after irradiation. Hematoxylin-and-eosin-stained
photomicrographs of representative unirradiated mouse skin and
UV-irradiated skin 24 to 72 hours after exposure, shown in Figure 1
, revealed the presence of sunburn cells
that exhibited the classic feature of apoptosis, pyknotic nuclei. In
the unirradiated mouse skin, a few cells undergoing normal cell death
or differentiation were randomly distributed across the epidermis.
However, in UV-irradiated skin, there were many sunburn cells, the
number (59 ± 5 per 100 cells) peaking at 24 hours after
irradiation. Some of the sunburn cells at early time points after
irradiation had contracted nuclei and clear cytoplasm, whereas the
sunburn cells during the peak of this response (24 hours) had
contracted nuclei and pink cytoplasm, suggesting that they were more
likely related directly to UV-induced damage. In one case, at 48 hours
after UV, the entire epidermal outer layer showed marked parakeratosis
and many apoptotic nuclei (data not shown). In addition to formation of
sunburn cells, inflammatory cells were also observed in UV-irradiated
mouse skin. These ranged from a few dermal mononuclear cells in the
control skin to many mononuclear cells and neutrophil polymorphs
in the UV-irradiated skin. These cells were mainly focal in the dermis,
around blood vessels, and their numbers seemed to peak at 48 hours
after irradiation (data not shown).
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In addition to defining the sunburn cells histologically, the
UV-irradiated mouse skin was examined for the presence of
TUNEL-positive cells. Immunofluorescence pictures of representative
unirradiated mouse skin and UV-irradiated skin 24 to 72 hours after
irradiation, shown in Figure 2
, indicated
a gradual increase in apoptotic cells with increasing time after UV
irradiation. Although a few TUNEL-positive cells were observed in
unirradiated skin, the number of apoptotic cells in the epidermis
dramatically increased and peaked at 24 hours (68 ± 4 per 100
cells), then significantly decreased at 72 hours (4 ± 0.08 per
100 cells) after UV exposure. Interestingly, apoptotic cells were also
present in the dermis of UV-irradiated skin, suggesting that UV
radiation had penetrated and damaged the cells in the dermis.
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The time course for induction of p53 and
p21Waf1/Cip1 in UV-irradiated mouse epidermis was
first investigated by Western blot analysis with specific antibodies.
Western blot data of representative unirradiated mouse skin and
UV-irradiated skin 3 to 72 hours after irradiation is shown in Figure 3
. Maximum induction of p53 occurred 12
hours after UV (20.4 ± 2.3-fold) but declined to basal levels at
72 hours (0.005 ± 0.003-fold). Similarly,
p21Waf1/Cip1 expression closely followed p53
expression, reaching maximal levels 24 hours (5.6 ± 0.9-fold)
after UV irradiation.
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. Localization of p53, p21Waf1/Cip1, bax, and bcl-2 Expression in UV-Irradiated Mouse Skin
Immunohistochemical analysis was then used to
determine whether UV irradiation had any effect on the cellular
distribution of p53 and p21Waf1/Cip1, bax, and
bcl-2 expression at different time points after UV irradiation.
Although no positive signal for p53 was detected in unirradiated skin
(Figure 4)
, intense p53 nuclear
immunostaining was observed along the basal layer of the epidermis 12
hours after UV exposure (Figure 4)
and also appeared to
accumulate in the hair follicles. However, at 24 hours after
irradiation, p53 was expressed in both the basal and the suprabasal
layers. In addition, analogous to the presence of apoptotic cells in
the dermis (Figure 2)
, cells in the dermis also expressed high levels
of nuclear p53 immunostaining 12 to 24 hours after UV irradiation
(Figure 4)
, suggesting further that UV radiation had indeed penetrated
and damaged the cells in the dermis. Although the
p21Waf1/Cip1 expression was very low in the
epidermis of unirradiated skin, it was expressed at high levels,
peaking at 24 hours, in and around the nuclei in the upper layers of
the epidermis (mature and differentiated cells) in irradiated skin
(Figure 5)
. On the other hand, bax
immunoreactivity was both perinuclear and cytoplasmic and was high in
the upper layers of the epidermis 24 hours after UV (Figure 5)
. In
contrast to bax immunoreactivity, bcl-2 immunoreactivity was high in
unirradiated skin but decreased gradually over time after UV exposure
(Figure 5)
.
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Histological examination of UV-irradiated skin 3 to 72 hours after
exposure revealed a gradual increase in epidermal hyperplasia in
UV-irradiated skin. Focal hyperplasia of the skin occurred as early as
6 hours after irradiation when the epidermis was 4 to 6 cells thick
(data not shown); the hyperplasia became more uniform and was maximal
(about 14 to 18 cells thick) 48 to 72 hours after irradiation (Figure 1)
. In addition, epidermal hyperplasia was accompanied by increased
keratin production at 24 hours and hyperkeratosis at 48 and 72 hours
after irradiation. At 24 hours, cells in focal areas of the epidermis
were quite swollen and had fine granular cytoplasm.
To determine whether the cells in hyperplastic epidermis were
proliferating, the skin of UV-irradiated mice was analyzed for PCNA
expression, a marker of proliferation, by Western blot and
immunohistochemical analyses. The Western blot data shown in Figure 3
revealed that PCNA expression increased gradually, reaching maximal
levels at 48 hours (50.3 ± 5.0-fold) after irradiation and then
decreased at 72 hours after irradiation. At the cellular level,
although PCNA immunoreactivity was limited to the basal layer in
unirradiated skin, a strong nuclear immunostaining was detected
throughout the epidermis 48 hours after irradiation (Figure 4)
.
However, the number of PCNA-positive cells in the epidermis as well as
epidermal hyperplasia decreased to normal levels about 2 weeks
after UV irradiation (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, suggesting that the UV-induced DNA damage was excessive and was not
efficiently repaired, leading to activation of the apoptotic pathway,
which eliminates severely damaged cells. Several studies have shown
that p53 is involved in UV-induced apoptosis.15,36,44
Interestingly, wild-type p53 can simultaneously induce the genetic
programs of both G1 growth arrest and apoptosis
in the same cell type.45
Recently, Cotton and Spandau
showed that high doses of UV radiation induce human keratinocytes to
undergo apoptosis, whereas low doses do not but appear instead to
activate the repair of UV-induced DNA damage.26
The time course for induction of apoptosis by UV closely followed the
time course for induction of p53 and
p21Waf1/Cip1. In addition, UV-induced bax
expression was inversely correlated with bcl-2 expression (Figure 3)
.
These results suggest that activation of apoptosis by UV irradiation is
most likely mediated by the p53 pathway, which involves up-regulation
of bax and down-regulation of bcl-2. Our results on induction of p53
expression and its nuclear accumulation in response to UV irradiation
are consistent with previous reports both in
vitro20,22,23,26,27
and in
vivo.18,19,25
Interestingly, immunohistochemical
examination of p53 expression in human skin exposed to equally
erythemogenic doses of UVC (200–280 nm), UVB, and UVA (320–400 nm)
showed that UVA-induced p53 expression is confined to the innermost
basal layer of the epidermis and UVC-induced p53 expression to the
granular and stratum spinosum layers, whereas UVB-induced p53
expression, which is probably the most relevant to human skin cancer,
occurs uniformly through all layers of the epidermis.18
Because the Kodacel-filtered FS40 sunlamps we used emitted 53% UVB and
47% UVA and nuclear expression of p53 was found in both the basal and
suprabasal layers of the epidermis and in the dermis, we can conclude
that both UVA and UVB were responsible for induction of p53 in hairless
mouse skin. In addition, the presence of apoptotic cells in the dermis
(Figure 2)
further suggests that both UVB and UVA play a role in
induction of p53 and apoptosis.
As mentioned before, p53 also plays a key role in the regulation of
cell cycle events.46
Indeed, when p53 protein expression
is elevated, it turns on the transcription of one of its important
downstream genes,
p21Waf1/Cip1.22,29-31
The
p21Waf1/Cip1 protein subsequently binds and
inhibits cyclin-dependent kinases, preventing phosphorylation of
critical cyclin-dependent kinase substrates and blocking cell cycle
progression, presumably to provide extra time for the cell to repair
DNA damage. Our findings that UV induction of p53 preceded induction of
p21Waf1/Cip1 protein (Figure 3)
suggest that UV
induces p53, which, in turn, transactivates
p21Waf1/Cip1 and causes cell cycle arrest to
permit the repair of UV-induced DNA damage. This conclusion is
supported by studies of Liu and Pelling,47
who
demonstrated that UV induction of p21Waf1/Cip1 in
mouse keratinocytes is mediated by p53 because UV irradiation does not
induce p21Waf1/Cip1 in p53-deficient cells.
Contrarily, a recent study has shown that UV can induce p53-independent
p21Waf1/Cip1 protein expression in mouse keratinocytes
in vivo and in vitro.48
In addition,
UV-induced p21Waf1/Cip1 expression was confined
mainly to the upper layer of the epidermis, further supporting a role
for p21Waf1/Cip1 protein as a marker of
terminally differentiated keratinocytes.49
In normal human and mouse epidermis, cells are constantly turning over;
stem cells divide and generate into keratinocytes that differentiate
and desquamate on the surface of the skin. Thus, differentiated cells
are constantly replaced by proliferating cells from the basal layer.
PCNA, a subunit of DNA polymerase 
, is known to play a role in DNA
replication and repair and serves as a biomarker of proliferation. It
is interesting to note that DNA-damaging agents induce both p53 and
PCNA expression.19,21,28
More importantly, the wild-type
53 protein is known to transcriptionally activate the PCNA
promoter,50
providing a mechanism whereby p53 induces PCNA
expression as a cellular response to DNA damage. On the other hand,
p21Waf1/Cip1 can inhibit cell cycle progression
by forming a quaternary complex with cyclin, cyclin-dependent kinase,
and PCNA.45
In our study, the level of PCNA expression
increased dramatically and was maximal at 48 hours after irradiation
(Figure 3)
, and the PCNA-positive cells were seen throughout the basal
and suprabasal layers of the skin (Figure 4)
and coincided with the
increase in hyperplasia (Figure 1)
. This observation is consistent with
the results of a previous study43
and suggests that UV
also activates a proliferative pathway, probably mediated by secretion
of growth factors in the skin, to replace apoptotic cells.
In summary, our results demonstrate that exposure of mouse skin to a single dose of UV results in the coordinated induction of p53, p21Waf1/Cip1, and bax as well as the formation of sunburn (apoptotic) cells. In addition, UV causes severe hyperplasia in the skin to replace the dead cells, suggesting that the processes of apoptosis and proliferation are closely linked and tightly regulated and that chronic UV irradiation may uncouple and dysregulate these two events, leading to the development of skin cancer.
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Acknowledgements |
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Footnotes |
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Supported by National Cancer Institute grants CA 46523 (to H. N. A.) and CA 16672 (institutional core grant). A. O. was supported by a McCarthy postdoctoral fellowship.
Accepted for publication September 15, 1999.
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